For species which use divergent chromosomes (such as X and Y) to determine sex, equalization of sex chromosome gene expression is the most immediate and vital aspect of sexual differentiation. This is termed dosage compensation, and in Drosophila it is achieved by a two- fold up regulation of X-linked genes in the male. The large, non-coding roX1 and roX2 RNAs (RNA on the X) share features suggesting a role in this process, the most striking of which is that both transcripts bind along the length of the male X chromosome. The long term objective of this project is to understand the mechanism by which a chromosome is selectively compensated. Proposed experiments focus on the role of roX RNAs. Initially the genetic interactions between roX1 and roX2, and between these genes and the male-specific lethals (msls), which are required for compensation, will be determined (Specific Aim I). The roX genes are positively regulated by the msls, revealing a previously unknown aspect of the msls function. Regulation of the roX genes will be further explored in Specific Aim II. The products of the msls bind to the male X chromosome as a ribonucleoprotein complex. In Specific Aim III the RNA component of the msls complex, which may include known roX RNAs, will be characterized. Specific Aims III and IV initiate a search for new members of the roX gene family. This information is vital to understanding the function of these RNAs. In contrast to Drosophila, compensation in humans involves inactivation of one of the female X chromosomes. However, both organisms appear to use similar mechanisms, such as histone acetylation, to modify transcription. Additionally, both species selectively coat the modified chromosome with RNA. The large Xist transcript binds to the length of the inactivated X in mammalian females, and is essential for the process of inactivation. These similarities suggest that understanding the mechanism of dosage compensation in Drosophila has relevance to other systems of global transcriptional regulation.